The inner ear is capable of mechanical detection at sub-nanometer scale, while displaying remarkable dynamic range. If the system could modulate its sensitivity of detection in response to the acoustic surroundings, it could reduce its gain of amplification or de-tune its frequency selectivity upon strong external stimulus, thus protecting itself from damage. Subsequent recovery to the original state would maintain optimal sensitivity of operation. The goal of the proposed research is to determine whether self-tuning occurs already at the sensory level, and determine its impact on the responsiveness of hair cells. Theoretical models have proposed that a hair cell contains an internal control parameter that determines its sensitivity of detection. Modulation of this parameter would control the gain of active amplification, and could cause the cell to exhibit instability by oscillating spontaneously. We aim to experimentally demonstrate crossing of this proposed bifurcation and explore potential biological mechanisms behind this phenomenon. Prolonged high-amplitude displacements will be applied to individual cells to mimic the effects of acoustic over-stimulation. Changes in active oscillation profiles and sensitivity of detection will be observed in real time, and the subsequent recovery recorded. Experimental demonstration of self-tuning of the mechanical response will allow us to establish a closer connection between the mathematical modeling and biologically relevant phenomena. In the first set of experiments, we will use mechanical manipulation to induce self-tuning in hair cells. The subsequent measurements will explore cellular mechanisms whereby a hair cell could self-adjust an internal parameter and thus modulate its dynamic state. Two potential biological parameters that we propose as control knobs for self-tuning in the hair cell are somatic membrane potential and internal calcium level. Simultaneous electrophysiological recordings and mechanical motility measurements will allow us to determine how the system of somatic ion channels interacts with the active mechanical amplifier. Sensitivity, frequency selectivity, and gain will be measured at different voltage-clamped levels. Pharmacological manipulation will be used to interfere with elements of the somatic circuit, and thus probe its impact on mechanical response. The effects of calcium on active bundle motility will be probed at multiple timescales. Fluorescence imaging will be combined with mechanical manipulation to extract the dynamics of calcium influx, accumulation, and extrusion from the stereocilia. Custom-designed instrumentation will be constructed to improve the temporal resolution currently accessible by confocal fluorescent imaging. The new technology will enable us to directly observe calcium signaling in biologically functional hair cells.